The invention relates to modified forms of single chain glycoprotein hormones wherein one or more cystine bonds in the xcex1 and/or xcex2 chain is disrupted. The cystine depleted forms are agonists or antagonists of the receptors for the relevant glycoprotein hormones.
The glycoprotein hormones, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG) are heterodimers composed of an xcex1 subunit, which is identical for all four hormones in a particular species, and xcex2 subunits which differ within a species according to the identity of the hormone. These hormones are found in all mammals although chorionic gonadotropin has been determined as present only in primates and equine species. Each of the xcex1 and xcex2 subunits contain multiple cystine bridges which appear necessary to effect the proper conformation for dimerization and secretion.
Recently, single chain forms of these glycoprotein hormones have been disclosed. PCT Application at WO 96/05224 published Feb. 22, 1996, and PCT Application WO 95/22304 published Aug. 24, 1995, describe various single chain forms of these hormones and recombinant methods for their production. See also Sugahara, T. et al. Proc Natl Acad Sci USA (1995) 92:201-2045; Narayan, P. et al., Mol Endocrinol (1995) 9:172-1726. In these single chain forms, the xcex1 and xcex2 portions are provided as a single fusion protein, optionally containing a linker, wherein either the xcex1 or xcex2 is at the N-terminus.
Cysteine depleted mutants of the xcex1 and xcex2 subunits and of the heterodimers of some of the human glycoprotein hormones have been reported previously. It has been shown that chemical reduction or site specific mutations of individual cysteine residues in either the common xcex1 or xcex2 subunit alters dimer assembly and secretion rate. Suganuma, N. et al, J Biol Chem (1989) 264:19302-19307 and Furuhashi, M. et al, J Biol Chem (1994) 269:25543-25548. In the present work, it is shown that mutants lacking either the 7-31 or 59-87 bonds of the a subunit were secreted and that heterodimers containing the xcex1 7-31 mutation bound to the LH/hCG receptor with comparable affinity to wild type hCG, whereas the xcex159-87 mutant interacted with lower binding affinity. Mutants comprising the cystine knot (10-60, 28-82 and 32-84) were not secreted in sufficient quantities to permit biological activity to be determined. The xcex17-31 and xcex159-87 forms had previously been prepared by Furuhashi, M., et a., supra.
Recently, the present applicant has reported the construction of xcex2xcex1 single-chain forms of chorionic gonadotropin wherein, in each such form, one of the six cystine bridges normally present in the xcex2 subunit has been deleted. These cystine depleted forms are still able to bind to the appropriate receptor and stimulate the production of cyclic AMP. (Ben-Menahem D. etal, J Biol Chem (1997)272: 6827-6830). Similarly, the present applicant as shown that deletion of single cystine bridges in the xcex1 chain portion of this xcex2xcex1 single chain hCG results in compounds with similar activity (Sato, A. et at., J Biol Chem (1997) 272:18098-18103).
The modified single-chain forms of the present invention provide additional members for the repertoire of agonists of the various heterodimeric glycoprotein hormones in various mammalian species and are useful as immunogens. Some members of this group may also act as antagonists.
Cystine bridge depleted single chain glycoprotein hormones derived from various species provide a new class of agonists (and antagonists) useful in enhancing (or inhibiting) fertility, modulating conditions associated with the reproductive system, and in the treatment of thyroid disorders. Enrichment of the resource of available agonists and antagonists provides added possibilities for treatment of individuals according to their unique metabolic and physiological profiles.
Thus, in one aspect, the invention is directed to a glycoprotein hormone agonist or antagonist which is a modified form of a glycoprotein hormone selected from the group consisting of follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid stimulating hormone (TSH) of the formula:
xcex1-(linker)nxe2x88x92xcex2xe2x80x83xe2x80x83(1)
or
(xcex2)xe2x88x92(linker)nxe2x88x92xcex1xe2x80x83xe2x80x83(2);
wherein xcex2 represents the xcex2 subunit of said FSH, LH, CG or TSH or a variant thereof; xcex1 represents the a subunit common to said glycoprotein hormones or a variant thereof; xe2x80x9clinkerxe2x80x9d represents an amino acid sequence providing a fusion protein between the C-terminus of a and the N-terminus of xcex2 in formula (1) or the C-terminus of xcex2 and the N-terminus of xcex1 in formula (2); and n is 0 or 1; and wherein one or more cystine bridges contained in said xcex1 and/or xcex2 subunit is deleted.
In other aspects, the invention is directed to recombinant materials and methods for the production of the agonists and antagonists of the invention, to pharmaceutical compositions containing these compounds, and to methods to modify the metabolism or physiology of a subject using the modified hormones of the invention.